Use of Salmonella / microsome reversion bioassay for ... · India (CPCB, 1990). In Rajasthan three such treatment plants have been established in Pali, ... Textile industries are

531

Journal of Environmental Biology ��May 2012��

Introduction

Small-scale industries, due to their limited resources in terms

of finance, space, and technology, cannot afford to treat their wastes

independently. Therefore Central Pollution Control Board (India) in

its comprehensive industry document series has advised setting up

of common effluent treatment plant (CETP) for small and medium

scale dye producing units.

A number of such plants are operational in different parts of

India (CPCB, 1990). In Rajasthan three such treatment plants have

been established in Pali, Jodhpur and Sumerpur to treat industrial

wastes mainly from textile mills. Out of these CETP, Pali has been

functional over a long period of time. This plant receives wastewater

effluents from 489 textile industries.

© 2012 Triveni Enterprises

Vikas Nagar, Lucknow, INDIA

[email protected]

Full paper available on: www.jeb.co.in

J. Environ. Biol.

33, 531-537 (2012)

ISSN: 0254-8704

CODEN: JEBIDP

Abstract

Salmonella / microsome reversion assay was used as a biological parameter for monitoring the toxicity

of common effluent treatment plant (CETP), Mandia road industrial area, Pali catering to textile industrial

areas in Pali, Rajasthan.The influent and effluent water of CETP, surface water (Bandi river) and under-

ground water were tested using Ames bioassay. The results showed presence of mutagens in surface

water of Bandi river and the underground water in Pali. Further, comparison of mutagenicity of CETP

influent and effluent water revealed that the treatment method employed at this plant has failed to remove

mutagenic substances present in Pali textile wastewater. The study also showed that Ames assay is an

important tool in genotoxic studies because of its simplicity, sensitivity to genetic damage, speed, low cost

of experimentation and small amount of sample required. Further Ames assay, as seen from the results of

this study, can be used as a monitoring tool for not only CETPs but also for other water resources. The

outcomes of the Ames assay demonstrated its performance as a sensitive, cost-effective and relatively

rapid screening tool to assess the genotoxic potential of complex environmental samples.

Key words

Common effluent treatment plant, Salmonella / microsome reversion assay, Textile effluents, Mutagenicity

Publication Data

Paper received:

03 November 2009

Revised received:

25 May 20010

Re-Revised received:

18 January 2011

Accepted:

16 May 2011

Use of Salmonella / microsome reversion bioassay for monitoring

industrial wastewater treatment plants in Rajasthan, India

Author Details

Nupur Mathur Environmental Toxicology Unit, Deptt. of Zoology, University of Rajasthan, Jaipur- 302 004, India

(Corresponding author) e-mail : [email protected]

Pradeep Bhatnagar Environmental Toxicology Unit, Deptt. of Zoology, University of Rajasthan, Jaipur- 302 004, India

Prakash Bakre Environmental Toxicology Unit, Deptt. of Zoology, University of Rajasthan, Jaipur- 302 004, India

Textile industries are known to discharge effluents containing

highly toxic compounds (Mathur et al., 2005; Mathur and Bhatnagar,2007; Hooda, 2007). Chhoakar et al. (2000) characterized theeffluents emanating from Pali textile mills and reported high salinity,

BOD (400-800 mgl-1) and COD (900-1500 mgl-1), excessiveconcentration of sodium and carbonate ions; high alkalinity (pH10.0- 11.5); and low concentrations of calcium in the textile effluents.The results of the study conducted by Mathur et al. (2005) clearlyindicate that most of the locally used dyes in Pali are highly mutagenic.The reason for this is that the textile industry is associated with

operations like bleaching, mercerizing, dyeing and printing whichutilize chemicals like sodium hypochlorite, H

2O2, acids, surfactants,

sodium hydroxide, dyestuffs urea, reducing agents, oxidizing agents,detergents and wetting agents resulting in high alkalinity, salinity,

dissolved solids, etc. Further, levels of heavy metals such as

532


Mathur et al.

Pb, Cr, Cu and Zn etc., in soils around the industrial area were

found to be significantly higher than their normal distribution in soil.

High concentrations of these toxic elements in soil were reported to

be responsible for the development of toxicity in agriculture products,

which in turn effects human life (Krishna and Govil, 2004). Thus, in

spite of the CETP, pollution problem in Pali continues to be grave.

Significant variations in the composition of the wastewater

arising from a cluster of industries have created difficulties in ensuring

the efficiency and effectiveness of the CETP. Besides, one of the

major drawbacks of the CETP is that the performance is usually

monitored only by physico-chemical parameters such as pH,

temperature, oil and grease, suspended solids, BOD and COD.

Consideration of only physico-chemical analysis has been thought

to be inadequate in protecting the aquatic environment against

hazardous discharges (Lambolez et al., 1994). Further such

analytical monitoring is not enough regarding the potential effects of

these effluents on human health. These parameters alone do not

reflect genotoxicity or other biological hazards of the effluents.

Further, in case of failure of CETP the entire untreated effluent be

released to environment. Proper monitoring of industrial treatment

plants is thus of utmost importance.

Microorganisms have demonstrated several attributes that

make them attractive for use in quick screening of effluents and chemicals

for toxicity. Testing of chemicals for mutagenicity in Ames assay is

based on the knowledge that a substance that is mutagenic in the

bacterium is likely to be a carcinogen in laboratory animals, and

thus,by extension, presents a risk of cancer to humans. The Ames

test has several advantages over the use of mammals for testing

compounds. It is relatively cost effective, only a few days are required

for testing a compound and the test is performed with microgram

quantities of the material. Such assays are performed on approximately

100 million organisms rather than on a limited number of animals.

Therefore to predict the additive, synergistic or antagonistic effect of

various chemicals on biological system, bioassay was used. In the

light of the above observations, the present work has been planned

to use short term microbial assay to monitor the genotoxicity of influent

and effluent water from industrial wastewater treatment plants. Further

the impact of these industrial wastes on genotoxicity of surface and

ground water of Pali was also investigated.

Materials and Methods

Study area: Pali, with a population of 18,19,200 people, is an

important district of Rajasthan. It is situated about 70 km from Jodhpur.

It has a geographical area of 12,387 sq km and is located between

24.45º to 26.75º N latitude and 72.48º to 74.20º E longitude. Pali,

situated on the banks of river Bandi, has got the largest number of

textile industries i.e. 989 in the state, mostly engaged in cotton and

synthetic textile printing and dyeing.

CETP chosen for the present study was the one installed at

Mandia road industrial area, Pali that caters to the need of about

489 textile industries located in this area. This plant treats only

industrial waste from these textile dyeing and printing industries.

The plant has a capacity to treat 1 million gallons per day (MGD) of

wastewater.

Common effluent treatment plant: The plant, developed by

Rajasthan Industrial Investment Corporation (RIICO), was

commissioned in the year 1986 (CPCB, 2005). National

Environment Engineering Research Institute (NEERI) prepared

the basic quality of influent and effluent waters. The monitoring

parameters are only physicochemical parameters i.e. BOD, COD,

pH and total suspended solids.

Sampling: Water samples were collected from five different

locations:

Underground water: Two samples were taken, first from a tube

well (TU) located in the residential area of Pali while second from

the boring water unit inside the effluent treatment plant (BU). Both

these sources were being used for drinking water purposes. Surface

water: Sample was taken from Bandi nallah drainage that ends up

in Bandi river (NL).

CETP: First sample was taken at the point where influent textile

wastewaters from the various textile industries at Mandia road, Pali,

are entering the treatment plant after passing through the grit

chamber (IF). Second sample was taken from the effluent water,

which was ready to be discharged into the river (EF). Samples

were collected in the month of April and October, which represent

the beginnings of summer and winter seasons, and were stored in

clean, sterile screw capped glass containers, at 4ºC.

Short- term microbial bioassay: Ames mutagenicity test: All the

water samples were tested in the ircrude natural state without

concentration The Salmonella / microsome reversion assay was

conducted using the plate incorporation procedure described by

Maron and Ames (1983). TA 98 and TA 100 strains of S. typhimurium

were obtained from microbial type culture collection and gene bank

(MTCC), Institute of Microbial Technology (IMTech), Chandigarh

(India).

The samples were analyzed with and without the hepatic

S9 fraction, which incorporates an important aspect of mammalian

metabolism into the in-vitro test. Samples were tested on duplicate

plates in two independent experiments. Five dose levels of individual

samples were tested. Positive controls used without metabolic

activation were 2-nitrofluorene for TA 98 (2.5 µg plate-1: 208

revertants) and sodium azide for TA 100 (5 µg plate-1: 2969

revertants). Positive control used with metabolic activation was 2-

anthramine for both TA 98 (1 µg plate-1: 481) and TA 100 (1 µg

plate-1 : 897). Sterile distilled water was used as negative control.

Fresh solutions of the reference mutagens were prepared

immediately before the beginning of each experiment. Sterile distilled

water was used as negative control (without metabolic activation: -

TA 98: 42 revertants and TA 100: 142 revertants; with metabolic

activation: TA 98: 44 revertants and TA 100: 168 revertants). All

533


Bioassay for monitoring industrial wastewater treatment plants

tester strains were maintained and stored according to the standard

methods (Mortelmans and Zeiger, 2000). The strains were regularly

checked for genetic markers. All reagents used were of analytical

grade, supplied by Himedia Laboratories Limited (India) and Sigma-

Aldrich (USA).

Statistical analysis: Multiple post-hoc comparison tests (LSD,

Tukey’s) were used for statistical analysis. A comparison-wise p-

value of <0.05 was considered statistically significant, and all tests

were two-tailed. Statistical Package for Social Sciences (SPSS),

release 10.0 was used for statistical analysis and graphical

representations (Saunders and Trapp, 1990).

Results and Discussion

A number of toxicological studies have been done using

algal, plant and animal models (Novotny et al., 2006; Muleyet al., 2007) including those on industrial wastewaters (Singh and

Singh, 2006; Soni et al., 2006). However, microorganisms have

demonstrated several attributes that make them attractive for use in

quick screening of effluents and chemicals for toxicity. The use of

bioassays is now an essential part of the hazard assessment and

control procedures of toxic chemicals and environmental mixtures.

In addition to their use in regulatory procedures, bioassays have

also been able to assess the performance of wastewater treatment

technologies (Jarvis et al., 2006; Alves et al., 2007).

Ames assay has been used extensively in the

genotoxicity testing of environmental contaminants. (Ohe et al.,

2004). Therefore, using Ames bioassay, influent and effluent

water of CETP were examined. Further using the same

genotoxicity-based criteria, surface and underground waters of

the area surrounding CETP were also analyzed. The results of

the Ames test for five different sampling sites are given in Table

1, as the mutagenicity ratio of average induced reversions

divided by spontaneous reversions. Mutagenicity ratio of 2.0 or

more is regarded as a significant indication of mutagenicity

provided all controls confirm to specifications (Mortelmans and

Zeiger, 2000). The mutagenicity tests were made both with and

without metabolic activation.

As seen from the mutagenicity ratios, both underground

water samples, taken from boring unit and Industrial tube well showed

ratios of more than 2.0 indicating positive mutagenicity with strain TA

98 (Table 1). During both the years, there was no significant

difference observed in the mutagenic activity in the months of April

and October. However, with strain TA 100, the boring water showed

Table 1 : Mutagenicity ratios of waters from CETP and industrial area, Pali with Salmonella tester strains TA 98 and TA 100

Mutagenicity ratio

Pali Site Vol. April October

(ml) TA 98 TA 100 TA 98 TA 100

- S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9

Boring unit (BU) 2 - + - - - + - +

5 + + - - - + - +

10 + + - - + + - +

50 + + - + + + - +

100 + + - + + + - +

Industrial tube well (TU) 2 - + - - - + - +

5 + + - - + + - +

10 + + - - + + - +

50 + + + + + + - +

100 + + + + + + - +

Drain (NL) 2 + + + - + + - +

5 + + + + + + - +

10 + + + + + + + +

50 + + + + + + + +

100 + + + + + + + +

Influent CETP (IF) 2 + - - - + - - -

5 + - + - + - + -

10 + + + - + + + +

50 + + + + + + + +

100 + + + + + + + +

Effluent CETP (EF) 2 + - - - + - + -

5 + + + - + - + -

10 + + + + + - + +

50 + + + + + + + +

100 + + + + + + + +

+ = Ratio greater than 2.0 indicating mutagenicity, - = Ratio less than 2.0 indicating non- mutagenicity

534


complete absence of mutagenicity in the months of April and

October.

The second under ground water sample, taken from an

industrial tube well, also showed complete absence of mutagenicity

during the month of October while during April, positive mutagenicity

was seen only at higher doses. Absence of mutagenic response

with strain TA 100 is, therefore, indicating absence of base-pair

substitution mutagens in these waters. Interestingly, the addition of

the hepatic fraction increased the number of revertants, indicating

Fig. 1: Dose response curve of water samples of Pali with strain TA 98 of Salmonella typhimurium (April); Error bars show 95.0% Cl of mean

Fig. 2: Dose response curve of water samples of Pali with strain TA 98 of Salmonella typhimurium fraction (October); Error bars show 95.0% Cl of mean

that, mammalian enzymes are possibly converting some of the

promutagenic compounds into active mutagenic metabolites.

Further with the surface waters of Nallah drainage,

positive mutagenicity was seen from the mutagenicity ratios, for

both the strains during April and October and at almost all the

dose levels tested. In case of Bandi nallah drainage, the textile

industry effluents being discharged were having high mutagenic

activity and were mainly responsible for contamination of these

surface waters.

Mathur et al.

535


More detailed observations were made with the dose

response curves of the water samples of the Pali industrial area,

shown in Fig. 1 to 4. In case of underground water samples of

Pali, with strain TA 98, a clear dose dependent response was

obtained, both in presence and absence of S9 hepatic fraction.

However, the number of revertants obtained with strain TA 98

during the month of April for boring unit and industrial tube well

(345 and 350 induced revertants, per 100 µl of sample, in absence

of S9 hepatic fraction, respectively, Fig.1) were similar to those

observed during October (320 and 563 induced revertants, per

100 µl of sample, in absence of S9 hepatic fraction, respectively,

Fig.2) indicating no significant variation during the months of April

and October.

With strain TA 100, during the month of October, a dose

dependent increase in the number of induced revertants was

obtained but doubling of the number of spontaneous revertants

(330 induced revertants, per 100 µl of sterile, distilled water) was

not seen even at the highest dose levels tested (320 induced

revertants, per 100 µl of sample, Fig.4). When assayed with strain

TA 100, during the month of April, boring and tube well waters

showed no mutagenicity at lower doses, without S9 mix. However

Fig. 3: Dose response curve of water samples of Pali with strain TA 100 of Salmonella typhimurium (April); Error bars show 95.0% Cl of mean

Fig. 4: Dose response curve of water samples of Pali with strain TA 100 of Salmonella typhimurium (October); Error bars show 95.0% Cl of mean


536


weak, dose dependent mutagenic activity was observed in the

presence of S9 hepatic fraction (Fig.3).

Addition of S9 hepatic fraction to underground water samples

of Pali industrial area showed significant increase in the number of

revertants. With strain TA 98 during April, without S9 mix, boring

unit and industrial tube well water had lower number of revertants

(345 and 350 induced revertants, per 100 µl of sample, in absence

of S9 hepatic fraction, respectively, Fig.1) than with S9 mix or hepatic

fraction (628 and 588 induced revertants, per 100 µl of sample, in

presence of S9 hepatic fraction, respectively, (Fig.1).

The Bandi nallah drainage water showed high

mutagenicity as seen from the number of induced revertants obtained

with strain TA 98 and TA 100. No significant difference was observed

in the number of induced revertants obtained with strain TA 98

during the months of April (3211 induced revertants, per 100 µ l of

sample, in absence of S9 hepatic fraction, Fig.1) and October (3672

induced revertants, per 100 µl of sample, in absence of S9 hepatic

fraction, Fig.2). Addition of S9 hepatic fraction to the Nallah water

resulted in decrease in the number of revertants obtained. With

strain TA 98, lesser numbers of revertants were obtained with S9

hepatic mix, in the months of April and October (3004 and 3216

induced revertants, per 100 µ l of sample, in presence of S9 fraction,

respectively, than without S9 fraction (3211 and 3672 induced

revertants, per 100 µl of sample, in absence of S9 hepatic fraction,

respectively (Fig. 1, 2).

Mutagenic activity of wastewaters of Pali textile dyeing and

printing units entering CETP as influent wastewaters (3000-3200

induced TA 98 revertants, per 100 µl of sample, in the absence of

S9 liver preparation (Fig. 1, 2) indicates moderate mutagenicity.

These results are in agreement with mutagenicity rankings by Houk

(1992), which places textile dyeing and textile plant wastewaters in

the category of moderate mutagenicity. The net number of TA 98

and TA 100 revertants per liter, in absence of S9 fraction, for

wastewaters of Pali textile dyeing and printing units is of the order

106-107 which is almost a degree higher than those reported by

Houk (1992) for other textile industry effluents.

Besides the mutagenicity data of the influent wastewater at

CETP Pali was comparable to the Bandi drainage Nallah water

data. The specific activity of mutagens in the drainage water (3200-

3600 induced TA 98 revertants, per 100 µl of sample, in the absence

of S9 liver preparation) was of the same order of magnitude as that

of mutagens detected in influent wastewater of CETP (3000-3200

induced TA 98 revertants, per 100 µl of sample, in the absence of

S9 liver preparation) (Fig. 1, 2).

These results clearly indicate that the textile influent

wastewater entering the CETP and the wastewaters being directly

discharged in local drainage Nallah contain almost same levels of

mutagenic compounds. This is a clear indication that many of the

textile dye and printing industries of Pali district are still discharging

a large amount of untreated waste directly into local water bodies.

The overall mutagen concentrations in the final effluents

being discharged by CETP Pali were no less than those present in

the influent wastewater. Influent water of CETP when assayed in

the absence of S9 hepatic fraction in the months of April and October

(3113 and 3034 induced TA 98 revertants, per 100 µl of the sample,

in absence of S9 mix, respectively) showed revertants in the same

order of magnitude as the effluent water of CETP (3668 and 3860

induced TA 98 revertants, per 100 µl of sample, in absence of S9

hepatic fraction, respectively). On addition of liver enzyme fraction

S9 a comparable decrease in the numbers of revertants was

observed for influent water (1745 and 1007 induced revertants,

per 100 µl of sample, in, and 1511, induced revertants, per 100 µl

of sample, in presence of S9 hepatic fraction, respectively) of CETP,

Mandia road, Pali (Fig. 1, 2).

Comparison of the mutagenic response of CETP influent

and effluent samples thus revealed that the treatment method

employed at this plant has failed to remove certain mutagenic

substances, which are present in the Pali textile wastewaters.These

results are in agreement with several previous studies which have

shown that many of the conventional and advanced wastewater

treatment plants have been unsuccessful in adequately removing

potentially hazardous chemical mutagens from the wastewater (Rizzo

et al., 2009; Gartiser et al., 2010; Torres-Guzman et al., 2010).

Further Ames assay, as seen from the results of this study,

can be used as a monitoring tool for not only CETPs but also for

other water resources. The outcomes of the ames assay

demonstrated its performance as a sensitive, cost-effective and

relatively rapid screening tool to assess the genotoxic potential of

complex environmental samples.

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Documents

Use of Salmonella / microsome reversion bioassay for ... · India (CPCB, 1990). In Rajasthan three such treatment plants have been established in Pali, ... Textile industries are